Alan D. Schreiber

Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3′-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

Enterocyte TLR4 Mediates Phagocytosis and Translocation of Bacteria Across the Intestinal Barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcγRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.

Modulation of Rab5 and Rab7 Recruitment to Phagosomes by Phosphatidylinositol 3-Kinase

ABSTRACT Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.

Immune thrombocytopenia. Use of a Coombs antiglobulin test to detect IgG and C3 on platelets

We applied a radiolabeled Coombs antiglobulin test to the diagnosis and management of immune thrombocytopenia in adults and children. This assay substantiated that the majority of patients with idiopathic thrombocytopenic purpura have increased levels of IgG on their platelets. Platelet-associated C3 was elevated in a subset of these patients, some of whom had normal levels oet-associated IgG, thus suggesting a role for C3 in the pathogenesis of the thrombocytopenia. The assay enabled us to anticipate a change in clinical status, establish the presence of immunologic remission after splenectomy and propose several mechanisms by which corticosteroids act. Platelets from a patient with the post-transfusion-purpura syndrome also carried increased IgG, indicating a role for IgG antibody or IgG-containing immune complexes in the destruction of host platelets in this disease. The radiolabeled Coombs test provides a general means to help diagnose, manage and study immune platelet disorders.

A randomized clinical trial of granulocyte transfusions for infection in acute leukemia.

In a prospective, controlled, randomized study to evaluate the efficacy of filtration-leukapheresis granulocytes in granulocytopenic, febrile patients with leukemia, 19 patients received antibiotics alone, and 12 received antibiotics plus daily granulocyte transfusions from ABO-matched donors. In skin-chamber studies the granulocytes appeared at sites of inflammation for at least six hours after transfusion. Infected subjects survived longer if they received granulocytes. Differences between control and transfused patients were greatest in patients with persistent bone-marrow failure, the 21-day survival being 20 per cent in controls, and 75 per cent in transfused patients. Granulocytes appeared to have no effect on the outcome of febrile episodes in which infection was not documented, the 21-day survival being 79 per cent for controls and 88 per cent for transfused patients. The transfusion of granulocytes thus appears to offer a survival advantage to infected, persistently granulocytopenic patients.

Heparin-Associated Thrombocytopenia

We studied the mechanism of platelet injury in 20 patients receiving heparin, three of whom became thrombocytopenic. Platelets from these three patients had increased levels of IgG and C3, which correlated with the presence of thrombocytopenia; their plasma caused the release of serotonin from normal platelets at concentrations of heparin within the usual therapeutic range. This reaction required IgG and an intact classic complement pathway. The 17 patients receiving heparin in whom thrombocytopenia did not develop had normal levels of platelet-associated IgG and C3. Plasma from 14 of these patients caused the release of serotonin, but only at heparin concentrations above the therapeutic range, in a reaction that also required IgG and complement. The addition of heparin to 15 normal plasma samples did not cause platelet injury in vitro. These studies indicate that heparin administration can be associated with complement-mediated platlet injury. The dose-dependent nature of this process may account for the occurrence of thrombocytopenia in some of these patients.

Pathophysiology of Immune Hemolytic Anemia

Abstract Studies of the pathophysiology of autoimmune hemolytic anemia emphasize the important role of cell membrane receptors for various immunologically active proteins in the clearance of foreig...

Immunologic infertility: identification of patients with antisperm antibody.

Immunologic causes of infertility have been postulated for many years, but a precise means for identification of men or women with antibodies against sperm has not been available. We studied 614 patients, including 257 couples, with unexplained in fertility, to detect the presence of circulating antisperm antibody. Using a specific radiolabeled-antiglobulin test, we identified a subset of patients (10 per cent) with IgG antisperm antibody; this antibody was present in 13 per cent of the women and 7 per cent of the men. We followed the course of six infertile couples in whom one partner had sperm antibody. In four couples, pregnancy was achieved; pregnancy correlated with resolution of antibody activity. In two of these four couples the partner (one man and one woman) with antisperm activity had been pretreated with corticosteroids. We conclude that this objective test may be used to identify and then to help manage infertility in patients with suspected antibody-mediated infertility.

Impaired Function of Macrophage Fcγ Receptors and Bacterial Infection in Alcoholic Cirrhosis

Background Bacterial infection is a frequent and often fatal complication in patients with cirrhosis. Macrophages play an important part in the host defense against infection because their Fcγ receptors recognize antibody-coated bacteria. Methods We prospectively studied macrophage Fcγ-receptor function in vivo and in vitro in 49 patients with alcoholic cirrhosis, 10 alcoholics without cirrhosis, and 20 normal volunteers. Results The clearance of IgG-sensitized autologous red cells was decreased in 37 of the 49 patients with cirrhosis but in none of the subjects without cirrhosis. In the 49 patients clearance was inhibited by a mean (±SE) of 47 ±3 percent at 1 hour and 53 ±3 percent at 1 1/2 hours, as compared with the clearance in the normal controls (P<0.001). The impairment of macrophage Fcγ-receptor-dependent clearance correlated with the degree of liver insufficiency but not with age, sex, nutritional status, HLA haplotype, or the presence of circulating immune complexes. The clearance of unsensitize...

Inhibition by CāINH of Hageman Factor Fragment Activation of Coagulation, Fibrinolysis, and Kinin Generation

Highly purified inhibitor of the first component of complement (CāINH) was shown to inhibit the capacity of active Hageman factor fragments to initiate kinin generation, fibrinolysis, and coagulation. The inhibition of prealbumin Hageman factor fragments observed was dependent upon the time of interaction of the fragments with CāINH and not to an effect upon kallikrein or plasmin generated. The inhibition of the coagulant activity of the intermediate sized Hageman factor fragment by CāINH was not due to an effect on PTA or other clotting factors. The inhibition by CāINH of both the prealbumin and intermediate sized Hageman factor fragments occurred in a dose response fashion. The CāINH did not appear to be consumed when the activity of the Hageman factor fragments was blocked, although the fragments themselves could no longer be recovered functionally or as a protein on alkaline disc gel electrophoretic analysis. These results suggest that the CāINH may have an enzymatic effect on the fragments or that an additional site on CāINH is involved in Cā inactivation.